Materials and methods for treatment of inflammation

ABSTRACT

The subject invention pertains to peptides and salts thereof that are useful as anti-inflammatory agents and to compositions containing such peptides and salts as active ingredients. Specifically exemplified herein are endomorphin-1 peptide (EM-1), analogs and salts thereof, and uses for modulation of calcitonin gene-related peptide (CGRP) production and/or substance P (SP) and for treatment of inflammation, particularly neurogenic inflammation.

CROSS-REFERENCE TO RELATED APPLICATIONS

The subject application is a continuation of co-pending application Ser.No. 13/113,392, filed May 23, 2011; which claims the benefit of U.S.Provisional Application Ser. No. 61/347,102, filed May 21, 2010, whichare hereby incorporated by reference in their entirety, including anyfigures, sequences, and/or tables.

The Sequence Listing for this application is labeled“As-filed_ST25.txt”, which was created on May 23, 2011, and is 8 KB. Theentire content is incorporated herein by reference in its entirety.

BACKGROUND OF INVENTION

Field of the Invention

The subject invention relates to uses of the endomorphin-1 peptide,analogs and salts thereof for treating and alleviating inflammation, inparticular, neurogenic inflammation.

Description of the Related Art

Much research has been devoted to development of compounds havinganti-inflammatory properties. Although certain methods and chemicalcompositions have been developed that aid in inhibiting or controllinginflammation, improved anti-inflammatory methods and compositions areneeded.

Neuroinflammatory conditions are complex and poorly understood diseaseprocesses. Diseases or disorders associated with neurologicalinflammation include neurogenic inflammation, meningitis, septic shock,Down's syndrome, postischemic brain injury, HIV encephalopathy,Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosisand multiple sclerosis.

Present therapies for inflammatory conditions, particularlyneuroinflammation, are largely based on steroids and non-steroidalanti-inflammatory compositions. These compositions, however, are usuallyassociated with a high incidence of unsatisfactory toxicity and poorefficacy.

Three different types of opiate receptors have been found: delta (δ),kappa (κ) and mu (μ). Endomorphin-1 peptide (EM-1) and analogs have beenfound to exhibit opiate-like activity by binding to the mu (morphine)opiate receptor. The major putative function for opiates is their rolein alleviating pain. Other areas where opiates are well-suited for usein treatment are conditions relating to gastrointestinal disorders,schizophrenia, obesity, blood pressure, convulsions, and seizures. Thesepeptides, however, have not previously been reported to play any role ininflammatory processes.

BRIEF SUMMARY

The present invention provides novel and advantageous therapeuticmethods for treating inflammation, particularly neurogenic inflammation.The methods comprise administering, to a subject in need of suchtreatment, an effective amount of an isolated peptide or salt thereof,wherein the peptide has a general formula:Tyr-X₁-X₂-X₃,

wherein X₁ is Pro, D-Lys or D-Orn;

X₂ is Trp, Phe or N-alkyl-Phe, wherein alkyl has 1 to about 6 carbonatoms; and

X₃ is Phe, Phe-NH₂, D-Phe, D-Phe-NH₂ or p-Y-Phe, wherein Y is NO₂, F, Clor Br.

Advantageously, the methods of the present invention can be used tocontrol over-production of calcitonin gene-related peptide (CGRP) in asubject.

In another embodiment, the subject invention provides methods to controlover-production of Substance P (SP) in a subject.

The subject invention also provides pharmaceutical compositions, e.g.anti-neurogenic inflammatory compositions, containing as an activeingredient an effective amount, of one or more peptides according to theformula expressed above and a non-toxic, pharmaceutically-acceptablecarrier or diluent.

The pharmaceutical compositions of the subject invention can furthercomprise other active compounds. Such other active compounds include,but are not limited to, anti-inflammatory compounds such as, forexample, steroidal compounds, including hydrocortisone and the like; ornon-steroidal anti-inflammatories, including acetylsalicylic acid(aspirin), ibuprofen, acetaminophen, indomethacin, and the like. Theadditional active ingredient(s) can also be antiviral, antibacterial,antifungal or other antimicrobial compounds or antitumor compounds.

The methods of the present invention are useful for treating conditionsselected from, for example, inflammatory conditions associated withpain, osteoarthritis, inflammatory skin conditions, asthma,fibromyalgia, eczema, rosacea, migraine, psoriasis, intestinalinflammation, rheumatoid arthritis, neurogenic swelling, edema, bruises,burns, sunburn, meningitis, septic shock, allergy, and dermatitis.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 is a schematic drawing describing an experimental protocol inrats.

FIG. 2 is a schematic drawing describing an experimental protocol inrats.

FIG. 3 shows the effect of CYT-1010 on electrically-evoked release ofCGRP from isolated rat tracheae. Each column represents the mean±s.e.m.concentration of CGRP measured in the incubation medium of thepre-stimulated, stimulated and post-stimulated 8-min fractions of n=5experiments (5×2 tracheae). *P<0.05 (vs. respective fraction of thevehicle-treated control experiment) and ^(#)P<0.05, ^(##)P<0.01,^(###)P<0.001 (vs. pre-stimulated fraction of the respectiveexperiment). Statistical analysis was performed with one-way ANOVAfollowed by Dunnett post-hoc test.

FIG. 4 shows the effect of CYT-1010 on electrically-evoked release of SPfrom isolated rat tracheae. Each column represents the mean±s.e.m.concentration of SP measured in the incubation medium of theprestimulated, stimulated and poststimulated 8-min fractions of n=5experiments (5×2 tracheae). *P<0.05 (vs. respective fraction of thevehicle-treated control experiment) and ^(#)P<0.05, ^(##)P<0.01,^(###)P<0.001 (vs. prestimulated fraction of the respective experiment).Statistical analysis was performed with one-way ANOVA followed byDunnett post-hoc test.

FIG. 5 shows the concentration-response curve demonstrating theinhibitory effect of CYT-1010 (10-2000 nM) on electrically-evokedrelease of CGRP from isolated rat tracheae. Concentration of the peptidein the basal, pre-stimulated fraction was subtracted from both therespective stimulated and post-stimulated fractions and then thesevalues were added to calculate the absolute release. Data pointsrepresent the percentage inhibitory effects of CYT-1010 compared to thecontrol, vehicle-treated group (n=5).

FIG. 6 shows the concentration-response curve demonstrating theinhibitory effect of CYT-1010 (10-2000 nM) on electrically-evokedrelease of SP from isolated rat tracheae. Concentration of the peptidein the basal, prestimulated fraction was subtracted from both therespective stimulated and poststimulated fractions and then these valueswere added to calculate the absolute release. Data points represent thepercentage inhibitory effects of CYT-1010 compared to the control,vehicle-treated group, n=5.

FIG. 7 shows the effect of CYT-1010 on 5% mustard oil-induced neurogenicear swelling of the mouse. Effect of endomorphin-1 (EM-1; i.p.) on 1%mustard oil-induced neurogenic ear swelling of the mouse. In the controlgroup saline (solvent) was applied i.p. in the same volume. Each datapoint represents the mean of n=8-9 experiments±s.e.m. expressed in %swelling as compared to the solvent-treated control group. *P<0.05;**P<0.01 vs. vehicle-treated control (one-way ANOVA followed by Dunnetpost test).

FIG. 8 shows the effect of CYT-1010 on 1% mustard oil-induced plasmaprotein extravasation in the acutely denervated hindpaw skin of the rat.In the control group, the vehicle (20% HPCD) was applied i.v. in thesame volume. Each column shows the mean±s.e.m. of n=8 rats; ***<0.001vs. vehicle-treated control group (one-way ANOVA followed by Dunnettpost test); ^(##)P<0.01 vs. the 10 μg/kg dose, ⁺P<0.05 vs. the 1000μg/kg dose (one-way ANOVA followed by Bonferroni's t-test).

FIG. 9 shows the effect of non-filtered i.v. CYT-1010 solution on 5%mustard oil-induced neurogenic ear swelling of the mouse. In the controlgroup the 20% HPCD vehicle was applied i.v. in the same volume withoutfiltration. Each data point represents the mean of n=8-9 mice±s.e.m.expressed in % swelling as compared to the control group. *P<0.05;**P<0.01 vs. vehicle-treated control (one-way ANOVA followed byDunnett's post-test).

FIG. 10 shows the effect of filtered i.v. CYT-1010 solution on 5%mustard oil-induced neurogenic ear swelling of the mouse. In the controlgroup the 20% HPCD vehicle was applied i.v. in the same volume afterfiltration. Each data point represents the mean of n=8 mice±s.e.m.expressed in % swelling as compared to the control group. *P<0.05;**P<0.01 vs. vehicle-treated control (one-way ANOVA followed byDunnett's post-test).

BRIEF DESCRIPTION OF SEQUENCES

SEQ ID NO:1 is a peptide useful according to the subject invention.

SEQ ID NO:2 is a peptide useful according to the subject invention.

SEQ ID NO:3 is a peptide useful according to the subject invention.

SEQ ID NO:4 is a peptide useful according to the subject invention.

SEQ ID NO:5 is a peptide useful according to the subject invention.

SEQ ID NO:6 is a peptide useful according to the subject invention.

SEQ ID NO:7 is a peptide useful according to the subject invention.

SEQ ID NO:8 is a peptide useful according to the subject invention.

SEQ ID NO:9 is a peptide useful according to the subject invention.

SEQ ID NO:10 is a peptide useful according to the subject invention.

SEQ ID NO:11 is a peptide useful according to the subject invention.

SEQ ID NO:12 is a peptide useful according to the subject invention.

SEQ ID NOS:13-26 are additional peptides useful according to the subjectinvention.

DETAILED DISCLOSURE

The subject invention pertains to novel uses as anti-inflammatory agentsof endomorphin-1 peptide, analogs, and salts thereof. The peptides andcompositions of the present invention are particularly effective ininhibiting neurogenic inflammation.

Peptides

In preferred embodiments, the present invention pertains to the use ofpeptides that have the general formula: Tyr-X₁-X₂-X₃, wherein X₁ is Pro,D-Lys or D-Orn; X₂ is Trp, Phe or N-alkyl-Phe, wherein alkyl has 1 toabout 6 carbon atoms; and X₃ is Phe, Phe-NH₂, D-Phe, D-Phe-NH₂ orp-Y-Phe, wherein Y is NO₂, F, Cl or Br. Some preferred peptides of theinvention are:

(SEQ ID NO: 1) H-Tyr-Pro-Trp-Phe-NH₂ (SEQ ID NO: 2)H-Tyr-Pro-Phe-Phe-NH₂ (SEQ ID NO: 3) H-Tyr-Pro-Trp-Phe-OH (SEQ ID NO: 4)H-Tyr-Pro-Phe-Phe-OH (SEQ ID NO: 5) H-Tyr-Pro-Trp-D-Phe-NH₂(SEQ ID NO: 6) H-Tyr-Pro-Phe-D-Phe-NH₂ (SEQ ID NO: 7)H-Tyr-Pro-Trp-pNO₂-Phe-NH₂ (SEQ ID NO: 8) H-Tyr-Pro-Phe-pNO₂-Phe-NH₂(SEQ ID NO: 9) H-Tyr-Pro-N-Me-Phe-Phe-NH₂ (SEQ ID NO: 10)H-Tyr-Pro-N-Et-Phe-Phe-NH₂ (SEQ ID NO: 11) H-Tyr-Pro-N-Me-Phe-D-Phe-NH₂(SEQ ID NO: 12) H-Tyr-Pro-N-Et-Phe-D-Phe-NH2 (SEQ ID NO: 13)H-Tyr-c-[D-Lys-Trp-Phe] (SEQ ID NO: 14) H-Tyr-c-[D-Lys-Phe-Phe](SEQ ID NO: 15) H-Tyr-c-[D-Orn-Trp-Phe] (SEQ ID NO: 16)H-Tyr-c-[D-Orn-Phe-Phe] (SEQ ID NO: 17) fl-Tyr-c-[D-Lys-Trp-pNO₂-Phe](SEQ ID NO: 18) H-Tyr-c-[D-Lys-Phe-pNO₂-Phe] (SEQ ID NO: 19)H-Tyr-c-[D-Orn-Trp-pNO₂-Phe] (SEQ ID NO: 20)H-Tyr-c-[D-Orn-Phe-pNO₂-Phe] (SEQ ID NO: 21)H-Tyr-c-[D-Lys-N-Me-Phe-Phe] (SEQ ID NO: 22)H-Tyr-c-[D-Orn-N-Me-Phe-Phe] (SEQ ID NO: 23)H-Tyr-c-[D-Lys-N-Et-Phe-Phe] (SEQ ID NO: 24)H-Tyr-c-[D-Orn-N-Et-Phe-Phe] (SEQ ID NO: 25)H-Tyr-c-[D-Lys-N-Me-Phe-D-Phe] (SEQ ID NO: 26)H-Tyr-c-[D-Lys-N-Et-Phe-D-Phe]

The last fourteen peptides listed are cyclic peptides whose linearprimary amino acid sequences are given in SEQ ID NO:13 through SEQ IDNO:26. In this context, the applicants incorporate herein by reference,in its entirety, U.S. Pat. No. 6,303,578.

The peptide of SEQ ID NO:1 is highly selective and very potent for the.mu.opiate receptor, with over 4000-fold weaker binding to deltareceptors and over 15,000-fold weaker binding to kappa receptors,thereby reducing the chances of side-effects.

The peptides of this invention may be prepared by conventionalsolution-phase (Bodansky, M., Peptide Chemistry: A Practical Textbook,2^(nd) Edition, Springer-Verlag, New York (1993) or solid phase(Stewart, J. M.; Young, J. D. Solid Phase Peptide Synthesis, 2^(nd)edition, Pierce Chemical Company, 1984) methods with the use of properprotecting groups and coupling agents. A suitable deprotection methodmay then be employed to remove specified or all of the protectinggroups, including splitting off the resin if solid phase synthesis isapplied.

Cyclization of the linear peptides can be performed by, for example,substitution of an appropriate diamino carboxylic acid for Pro inposition 2 in the peptides through ring closure of the 2-position sidechain amino and the C-terminal carboxylic functional groups. Thecyclization reactions can be performed with the diphenylphosphoryl azidemethod (Schmidt, R., Neuhert, K., Int. J. Pept. Protein Res. 37:502-507,1991).

Peptides synthesized with solid phase synthesis can be split off theresin with liquid hydrogen fluoride (HF) in the presence of the properantioxidant and scavenger.

The desired products may be isolated from the reaction mixture bycrystallization, electrophoresis, extraction, chromatography, or othermeans. However, a preferred method of isolation is HPLC. All of thecrude peptides can be purified with preparative HPLC, and the purity ofthe peptides may be checked with analytical HPLC. Purities greater than95% of the synthesized compounds using HPLC have been obtained.

In a preferred embodiment specifically exemplified herein, the peptideis that which is shown as SEQ ID NO:13 (cyclic endomorphin-1 peptide)and has the following structure:

Surprisingly, it has now been discovered that endomorphin-1 peptide(EM-1) and analogs thereof can be used to modulate immune responses andalleviate inflammatory conditions, in particular, neurogenicinflammatory conditions and/or inflammatory conditions associated withpain. The peptides of the present invention are potent inhibitors ofcalcitonin gene-related peptide (CGRP) release in the peripheral and thecentral nervous systems. CGRP, expressed as CGRP-α and CGRP-β in humans,plays a pivotal role in autonomic, sensory, and motor activities via itsinteractions between the neurological, endocrine, and immune systems. Itfunctions as a potent vasodilator and mediates the transmission of painthroughout the body.

CGRP expression is regulated, in part, by the expression of variousimmunomodulators such as cytokines, tumor necrosis factor-α (TNF-α),iNOS, and mitogen-activated protein kinases (MAPK). In turn, CGRP isalso involved in the development and progression of inflammatoryreactions, such as the accumulation of cAMP, T cell proliferation, andthe secretion of interleukins such as IL-1 and IL-6 and cytokines inhuman blood monocytes. Serum concentrations of CGRP are markedlyelevated in inflammation, systematic infections, joint disorders, and avariety of other diseases such as cardiac failure and hypertension.Particularly, CGRP is involved in many acute and chronic inflammatoryreactions, such as for example, neurogenic inflammation, rheumatoidarthritis, sepsis, migraine, and endocrine diseases.

The release of CGRP from peptidergic afferents of isolated rat tracheaeis evoked by electrical field stimulation (EFS) and its concentrationcan be determined from the incubation medium with radioimmunoassay. Ithas been found that EFS-induced CGRP release is significantly reduced byadding peptides of the present invention into the organ bath, during andafter stimulation.

Furthermore, peptides of the present invention effectively suppressacute neurogenic inflammatory reactions in vivo. Neurogenic inflammationin the rat hindpaw skin and in the mouse ear is induced by activatingsensory fibres with topical application of mustard oil. Plasma proteinextravasation can be measured by Evans blue leakage and ear edema withmicrometry. Mustard oil-induced plasma extravasation in the rat skin wassignificantly decreased by i.v. pretreatment with 1-1000 μg/kg CYT-1010,the lowest dose exerted, almost 60% inhibitory action. A similarsignificant anti-edema effect was also found on the mouse ear for theseCYT-1010 doses throughout a 3-h period.

Treatment of Inflammation and Related Disorders

The peptides of the present invention, through administration to asubject, are useful for modulating immune responses and treatinginflammation, particularly, neurogenic inflammation. The term “subject,”as used herein, describes an organism, including mammals such asprimates, to which treatment with the compositions according to thepresent invention can be administered. Mammalian species that canbenefit from the disclosed methods of treatment include, but are notlimited to, apes, chimpanzees, orangutans, humans, monkeys; and otheranimals such as dogs, cats, horses, cattle, pigs, sheep, goats,chickens, mice, rats, guinea pigs, and hamsters

Advantageously, endomorphin-1 peptide and analogs thereof are powerfulagents for treatment of inflammatory responses, including neurogenicinflammatory responses, through the inhibition of sensory neuropeptiderelease. The peptides of the present invention provide effectivetreatment for neurological inflammatory conditions, where nociceptoractivation plays a pivotal role. At present, non-steroidalanti-inflammatory/analgesic drugs and corticosteroids are the mostextensively used pharmaceuticals for neurological inflammationassociated with nociceptor activation; however, these drugs, even inhigh doses, can only induce moderate inhibition of inflammatoryresponses.

In one embodiment, the present invention provides a method for treatinginflammatory and immune conditions. The method comprises administering,to a subject in need of such treatment, an effective amount ofendomorphin-1 peptide (EM-1), or an analog, and/or salt thereof. In aspecific embodiment, the present method treats or ameliorates acondition associated with, at least in part, neuroinflammation orneurogenic inflammation. In another embodiment, the present methodinhibits CGRP release, and is useful for treating, alleviating, orameliorating a disease or condition associated with abnormal CGRPactivity. In yet another embodiment, the methods of the subjectinvention can be used to inhibit SP release, and for treating a diseaseor condition associated with abnormal SP activity.

For purposes of the present invention, unless otherwise noted, the terms“inflammation” and “inflammatory response” include immune-relatedresponses and/or allergic reactions to a physical, chemical, orbiological stimulus. For example, inflammation for which the primaryactivating inflammation is antigen-derived can be due to, for example,bacterial lipopolysaccharide.

The term “neuroinflammation” or “neuroinflammatory diseases, disordersor conditions,” as used herein, includes any disease, disorder orcondition that is associated with the central and peripheral nervoussystems, including inflammation that occurs in response to brain injuryor autoimmune disorders, and inflammation that causes destruction ofhealthy neuronal and/or cerebral tissue.

“Neurogenic inflammation,” as used herein, includes the local release ofinflammatory mediators from afferent neurons such as substance P andcalcitonin gene-related peptide.

Particularly exemplified herein is the use of the peptides andcompositions of the present invention for treatment of neurogenicinflammation. Neurogenic inflammation can be evoked by neuropeptides,such as substance P (SP), calcitonin gene-related peptide (CGRP),vasoactive intestinal peptide (VIP), and neurokinin A (NKA), releasedfrom primary afferent C-fiber nerve terminals and histamine, secondarilyreleased from mast cells (Dray, A., [1992] “Neuro pharmacologicalmechanisms of capsaicin and related substances” Biochem Pharm44(4):611-15). In addition, it is known that capsaicin (CAP), the activeconstituent found in cayenne pepper, induces an acute neurogenicinflammatory response when applied topically to skin. CAP is a highlyselective pain-producing substance that selectively stimulatesnociceptive and thermal-sensitive nerve endings in tissues by acting ona specific membrane receptor. The mode of action of capsaicin,therefore, differs significantly from phorbol myristate acetate(PMA)-induced immune-inflammation. By comparison, PMA elicits itspro-inflammatory effects through cellular activation of specific immunecells such as macrophages.

The peptides and compositions of the present invention can be used totreat, alleviate, or ameliorate diseases and conditions associated withneurogenic inflammation including, but not limited to, osteoarthritis,asthma, fibromyalgia, eczema, rosacea, migraine, psoriasis, intestinalinflammation, rheumatoid arthritis, neurogenic swelling, edema, bruises,burns, sunburn, meningitis, septic shock, allergy, and dermatitis.

In addition, the peptides and compositions of the present invention canbe used to treat, alleviate, or ameliorate inflammation at sites wherethe primary activating factor is antigen-derived (e.g. bacteriallipopolysaccharide) or of neurogenic origin. In one embodiment, thepeptides of the subject invention are used to treat pathologicalinflammatory conditions of the brain.

In addition, the peptides and compositions of the present invention canbe used to treat, alleviate, or ameliorate a variety of inflammatoryskin conditions, in particular, skin conditions associated withinflammation and pain. The present invention can be used to treat,alleviate, or ameliorate inflammatory skin conditions including, but notlimited to, radiation irritation and burns (including UV and ionizing),chemical burns, rhinitis, thermal burns, reddening of the skin, andchemically induced lesions. Additionally, the present invention can beused to treat, alleviate, or ameliorate a variety of inflammatory skinconditions, such as for example, atopic dermatitis, dermatitis,psoriasis, lichen simplex, acne, eczema, professional dermatitis,seborrheic dermatitis, prurigo nodularis, urticaria, keratosis, rosacea,erythema, ichtyosis, photodermatoses, shingles, and pruritic skindisorders. Additionally, the present invention can be used to treat,alleviate, or ameliorate inflammatory skin conditions caused bymicro-organisms, chemicals, physical injury, environmental conditions,stress, aging, and autoimmune or inflammatory diseases. The peptides andcompositions of the present invention are particularly useful to treat,alleviate, or ameliorate skin conditions including psoriasis, allergiccontact dermatitis, eczema, urticaria, lichen planus, and dermatitisherpetiformis.

In addition, the peptides and compositions of the present invention canbe used to treat, alleviate, or ameliorate allergic responses. This caninclude the use of the peptides in aerosol form for the treatment ofacute allergic reactions such as acute asthmatic attack and in thetreatment of inflammation of the lung caused by chemical exposure.

The peptides and compositions of the present invention are particularlyuseful to treat, alleviate, or ameliorate diseases and conditionsassociated with pain and inflammation including, but not limited to,inflammatory joints, muscles, tendons, nerves and skin; osteo-arthritisand rheumatoid arthritis; dermatitis; inflammatory bowel disease;post-operative pain and inflammation; general blunt trauma; boneinjuries; soft tissue infections; and shingles.

In addition, the peptides and compositions of the present invention areparticularly useful to treat, alleviate, or ameliorate diseases andconditions, such as for example, asthma, fibromyalgia, eczema, rosacea,migraine, psoriasis, intestinal inflammation, rheumatoid arthritis,neurogenic swelling, edema, bruises, burns, sunburn, meningitis, septicshock, allergy, and dermatitis.

Therapeutic Compositions and Formulations

The present invention further provides therapeutic compositions thatcontain a therapeutically effective amount of the peptides or salts anda pharmaceutically acceptable carrier or adjuvant. The present inventionalso contemplates prodrugs or metabolites of the peptides.

As used herein, the terms “pharmaceutically acceptable”,“physiologically tolerable” and grammatical variations thereof, includecompositions, carriers, diluents and reagents, are used interchangeablyand represent that the materials are capable of administration to orupon a subject such as mammal.

The term “prodrug,” as used herein, refers to a metabolic precursor of acompound of the present invention or pharmaceutically acceptable formthereof. In general, a prodrug comprises a functional derivative of acompound, which may be inactive when administered to a subject, but isreadily convertible in vivo into an active metabolite compound.

Conventional procedures for the selection and preparation of suitableprodrug derivatives are described, for example, in “Design of Prodrugs”,ed. H. Bundgaard, Elsevier, 1985. Preferably, a prodrug of the presentinvention enhances desirable qualities of the compound of the presentinvention including, but not limited to, solubility, bioavailability,and stability. Hence, the compounds employed in the present methods may,if desired, be delivered in a prodrug form. Prodrugs of the compoundsemployed in the present invention may be prepared by modifyingfunctional groups present in the compound such that the modificationsare cleaved, either in routine manipulation or in vivo, to the parentcompound.

The term “metabolite,” refers to a pharmacologically active product,including for example, an active intermediate or an ultimate product,produced through in vivo metabolism of a compound of the presentinvention in a subject. A metabolite may result, for example, from theanabolic and/or catabolic processes of the administered compound in asubject, including but not limited to, the oxidation, reduction,hydrolysis, amidation, deamidation, esterification, deesterification,enzymatic cleavage, and the like.

Metabolites are typically identified by preparing a radiolabelled (e.g.,¹⁴C or .³H) isotope of a compound of the present invention,administering it parenterally in a detectable dose (e.g., greater thanabout 0.5 mg/kg) to an animal such as rat, mouse, guinea pig, monkey, orto a human, allowing sufficient time for metabolism to occur (typicallyabout 30 seconds to about 30 hours), and isolating its conversionproducts from the urine, blood or other biological samples. Theseproducts are easily isolated since they are labeled (others are isolatedby the use of antibodies capable of binding epitopes surviving in themetabolite). The structure of metabolites can be determined inconventional fashion, e.g., by MS, LC/MS or NMR analysis. In general,analysis of metabolites is performed according to techniques well knownto those skilled in the art of drug metabolism studies.

The peptide salts of the present invention may be compounded, forexample, with the usual non-toxic, pharmaceutically acceptable carriersfor tablets, pellets, capsules, liposomes, suppositories, intranasalsprays, solutions, emulsions, suspensions, aerosols, targeted chemicaldelivery systems (Prokai-Tatrai, K.; Prokai, L; Bodor, N., J. Med. Chem.39:4775-4782, 1991), and any other form suitable for use. The carrierswhich can be used are water, glucose, lactose, gum acacia, gelatin,mannitol, starch paste, magnesium trisilicate, talc, corn starch,keratin, colloidal silica, potato starch, urea and other carrierssuitable for use in manufacturing preparations, in solid, semisolid,liquid or aerosol form, and in addition auxiliary, stabilizing,thickening and coloring agents and perfumes may be used.

In one embodiment, acids suitable for preparing the peptide salts areshown in Table 1, and the corresponding peptide salts are shown in Table2. Preferred salt peptides include maleate salt, hydrochloride salt,lactate salt, aspartate salt, acetate salt, and trifluoro acetate salt.

TABLE 1 Acids Acetic Aspartic (L) Citric Fumaric Gluconic (D) HippuricHydrochloric Lactic Malic Maleic Mucic Phosphoric Sulfuric SuccinicTartaric (L)

TABLE 2 Salt Forms Acetate Hippurate Mucate Tartrate (L) Aspartate (L)Hydrochloride Phosphate Gluconate (D) Citrate Lactate Sulfate MaleateFumarate Malate (L) Hemi-sulfate Succinate

The peptide salts of the subject invention can also be used to provideanti-inflammatory treatments. In this context the applicants incorporateherein by reference, in its entirety, U.S. 2004/0266805.

Further, the therapeutic composition can comprise one or more peptidesor salts of the present invention as a first active ingredient, and oneor more additional active ingredients comprising an anti-inflammatorycompound known in the art. Such known anti-inflammatory drugs include,but are not limited to, steroidal anti-inflammatory drugs andnon-steroidal anti-inflammatory drugs (NSAIDs), includingacetylsalicylic acid (aspirin), ibuprofen, acetaminophen, indomethacin,and the like. The additional active ingredient(s) can be, for example,antiviral, antibacterial, antifungal or other antimicrobial compounds orantitumor compounds.

In accordance with one embodiment of the invention, therapeuticallyeffective amounts of a known anti-inflammatory agent and the peptides ofthe present invention are administered sequentially or concurrently to apatient. The most effective mode of administration and dosage regimen ofthe peptides of the present invention and anti-inflammatory agent willdepend upon the type of condition to be treated, the severity and courseof that condition, previous therapy, the patient's health status, andresponse to the peptides of the present invention and the judgment ofthe treating physician. The present compositions may be administered tothe patient at one time or over a series of treatments.

The present invention contemplates therapeutic compositions useful forpracticing the therapeutic methods described herein. Therapeuticcompositions of the present invention contain a physiologicallytolerable carrier together with a therapeutically effective amount of apeptide as described herein, dissolved or dispersed therein as an activeingredient.

The peptides used in these therapies can also be in a variety of forms.These include for example, solid, semi-solid and liquid dosage forms,such as tablets, pills, powders, liquid solutions or suspensions,suppositories, injectable and infusible solutions. The preferred formdepends on the intended mode of administration and therapeuticapplication.

The compositions also preferably include conventional pharmaceuticallyacceptable carriers and adjuvants which are known to those of skill inthe art. Preferably, the compositions of the invention are in the formof a unit dose and will usually be administered to the patient one ormore times a day.

The present peptides and compositions can be in a form that can becombined with a pharmaceutically acceptable carrier. In this context,the compound may be, for example, isolated or substantially pure. Theterm “carrier,” as used herein, refers to a diluent, adjuvant,excipient, or vehicle with which the compound is administered. Suchpharmaceutical carriers can be sterile liquids, such as water and oils,including those of petroleum oil such as mineral oil, vegetable oil suchas peanut oil, soybean oil, and sesame oil, animal oil, or oil ofsynthetic origin. Saline solutions and aqueous dextrose and glycerolsolutions can also be employed as liquid carriers, particularly forinjectable solutions. Particularly preferred pharmaceutical carriers fortreatment of or amelioration of inflammation in the central nervoussystem are carriers that can penetrate the blood/brain barrier. As usedherein carriers do not include the natural plants as they exist innature.

The amount of active ingredient that may be combined with the carriermaterials to produce a single dosage form will vary, depending such asthe type of the condition and the subject to be treated. In general, atherapeutic composition contains from about 5% to about 95% activeingredient (w/w). More specifically, a therapeutic composition containsfrom about 20% (w/w) to about 80% or about 30% to about 70% activeingredient (w/w).

The peptides of the present invention can be formulated according toknown methods for preparing pharmaceutically useful compositions.Formulations are described in detail in a number of sources which arewell known and readily available to those skilled in the art. Forexample, Remington's Pharmaceutical Science by E. W. Martin describesformulations which can be used in connection with the subject invention.In general, the compositions of the subject invention will be formulatedsuch that an effective amount of the bioactive compound(s) is combinedwith a suitable carrier in order to facilitate effective administrationof the composition.

The preparation of a pharmacological composition that contains activeingredients dissolved or dispersed therein is well understood in the artand need not be limited based on formulation. Typically suchcompositions are prepared as injectables either as liquid solutions orsuspensions; however, solid forms suitable for solution, or suspensions,in liquid prior to use also can be prepared. The preparation also can beemulsified.

The active ingredient can be mixed with excipients which arepharmaceutically acceptable and compatible with the active ingredientand in amounts suitable for use in the therapeutic methods describedherein. Suitable excipients are, for example, water, saline,dimethylsulphoxyde (DMSO) cyclodextrins, dextrose, glycerol, ethanol,sucrose, glucose, mannitol, sorbitol or the like and combinationsthereof. In addition, if desired, the composition can contain minoramounts of auxiliary substances such as wetting or emulsifying agents,pH buffering agents and the like which enhance the effectiveness of theactive ingredient. Particularly preferred excipients for peptides andcompositions of the present invention include dimethylsulphoxyde (DMSO),and hydroxypropyl-β-cyclodextrin.

Liquid compositions also can contain liquid phases in addition to and tothe exclusion of water. Exemplary of such additional liquid phases areglycerin, vegetable oils such as cottonseed oil, and water-oilemulsions.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients, e.g.,compound, carrier suitable for administration.

Routes of Administration

The peptides and compositions of the present invention can beadministered to the subject being treated by standard routes, includingthe oral, ophthalmic nasal, topical, transdermal, intra-articular,parenteral (e.g., intravenous, intraperitoneal, intradermal,subcutaneous or intramuscular), intracranial, intracerebral,intraspinal, intravaginal, intrauterine, or rectal route. Depending onthe condition being treated, one route may be preferred over others,which can be determined by those skilled in the art.

For instance, the peptides and compositions of the present invention canbe topically administered to the subject for treatment of conditionsassociated with skin inflammation. Compositions for topicaladministration can be in any of a variety of forms, includingsuspension, dispersion, solution, ointment, gel, cream, spray, foam,powder, lotion, soak, transdermal patch, solid, micro-particle, vapor,or tape.

The peptides of the present invention may also be administered utilizingliposome technology, slow release capsules, implantable pumps, andbiodegradable containers. These delivery methods can, advantageously,provide a uniform dosage over an extended period of time. The amount ofthe therapeutic composition of the invention which is effective in thetreatment of a particular disease, condition or disorder will depend onthe nature of the disease, condition or disorder and can be determinedby standard clinical techniques.

The dosage of effective amount of the peptides varies from and alsodepends upon the age and condition of each individual patient to betreated. In general, suitable unit dosages may be between about 0.01 toabout 500 mg, about 0.01 to about 400 mg, about 0.01 to about 300 mg,about 0.01 to about 200 mg, about 0.01 to about 100 mg, or about 0.01 toabout 50 mg. For example, a unit dose may be from between about 0.2 mgto about 50 mg. Such a unit dose may be administered more than once aday, e.g. two or three times a day.

In addition, in vitro assays may optionally be employed to help identifyoptimal dosage ranges. The precise dose to be employed in theformulation will also depend on the route of administration, and theseriousness of the disease, condition or disorder, and should be decidedaccording to the judgment of the practitioner and each patient'scircumstances. Effective doses may be extrapolated from dose-responsecurves derived from in vitro or animal model test systems.

Illustratively, dosage levels of the administered active ingredients canbe: intravenous, 0.01 to about 20 mg/kg; intraperitoneal, 0.01 to about100 mg/kg; subcutaneous, 0.01 to about 100 mg/kg; intramuscular, 0.01 toabout 100 mg/kg; orally 0.01 to about 200 mg/kg and preferably about 1to 100 mg/kg; intranasal instillation, 0.01 to about 20 mg/kg; andaerosol, 0.01 to about 20 mg/kg of animal (body) weight.

Once improvement of the patient's condition has occurred, a maintenancedose is administered if necessary. Subsequently, the dosage or thefrequency of administration, or both, may be reduced as a function ofthe symptoms to a level at which the improved condition is retained.When the symptoms have been alleviated to the desired level, treatmentshould cease. Patients may however require intermittent treatment on along-term basis upon any recurrence of disease symptoms.

In one embodiment, the peptides of the present invention and any secondanti-inflammatory agent are administered sequentially to the patient,with the anti-inflammatory agent being administered before, after, orboth before and after treatment with the peptides of the presentinvention. Sequential administration involves treatment with theanti-inflammatory agent at least on the same day (within 24 hours) oftreatment with peptides of the present invention and may involvecontinued treatment with the anti-inflammatory agent on days that thepeptides of the present invention is not administered.

Conventional modes of administration and standard dosage regimens ofanti-inflammatory agents may be used (see Gilman, A. G. et. al. [eds]The Pharmacological Basis of Therapeutics, pp. 697-713, 1482, 1489-1491[1980]; Physicians Desk Reference, 1985 Edition). For example,indomethacin can be administered orally at a dosage of about 25-50 mg,three times a day. Higher doses can also be used. Alternatively, aspirin(about 1500-2000 mg/day), ibuprofen (about 1200-3200 mg/day), orconventional therapeutic doses of other anti-inflammatory agents can beused. Dosages of anti-inflammatory agents can be titrated to theindividual patient.

In addition, the patient may receive concurrent treatments with theanti-inflammatory agents and compositions comprising the peptide of thepresent invention. For example, the peptide of the present invention canbe administered via local intralesional, or intravenous injection (seeGilman et. al. supra at pp. 1290-91). The anti-inflammatory agent canalso administered by subcutaneous injection, intravenous injection, ororally.

Alternatively, the patient can receive a composition comprising acombination of one or more peptides of the present invention and ananti-inflammatory agent according to conventional modes ofadministration of agents which exhibit antibacterial, anticancer,antitumor or anti-inflammatory activity. These include, for example,parenteral, subcutaneous, intravenous, or intralesional routes ofadministration.

Materials and Methods

CYT-1010 (synthetic cyclized endomorphin-1 analog; FP014; Lot #:080811-R2; Bottle H22; MW:684), was purchased from AmbioPharm, Inc.(North Augusta, S.C. 29812). Additional materials include:2-hydroxypropyl-βcyclodextrin (HPCD; CY2005.5; CYL-3122), purchased fromCyclolab Ltd., Budapest, Hungary; Na-thiopental (Thiopental-Sandoz),purchased from Sandoz, Kundl, Austria; urethane, purchased from Spektrum3D, Debrecen, Hungary; ketamine (Calypsol), purchased fromRichter-Gedeon Ltd., Budapest, Hungary; xylazine (Xylavet), purchasedfrom Phylaxia-Sanofi, Veterinary Biology Co. Ltd., Budapest, Hungary;rat α-CGRP, Tyr-α-CGRP(23-37), purchased from Bachem, Bubendorf,Switzerland; ¹²⁵I-labelled SP, purchased from Amersham, International,Amersham, UK; ¹²⁵I-labelled Tyr-α-CGRP(23-37), prepared in thelaboratory of the Department of Pharmacology and Pharmacotherapy,University of Pécs, Hungary; allylisothiocyanate (mustard oil), Evansblue dye, purchased from Sigma, St. Louis, Mo., USA; paraffin oil,formamide, dimethylsulphoxyde (DMSO), purchased from Szkarabeusz Ltd.,Pécs, Hungary.

Preparation of Solutions and Suspensions

For the in vitro release experiments, a 10 mg/ml (14.62 mM; MW 684)stock solution of CYT-1010 was made in DMSO, which was further dilutedwith the oxygenated Krebs solution used in the organ bath: 5.5 μl ofthis CYT-1010 stock solution was added into 40 ml Krebs to make thehighest applied 2 μM concentration of compound CYT-1010. Furtherdilutions to obtain the lower concentrations were prepared with Krebssolution.

The incubation media containing the respective CYT-1010 concentrationswere administered in the stimulated and post-stimulated 8-min fractions.

Since even the highest, 2 μM, concentration of CYT-1010 solutioncontained negligible amount of DMSO, simple Krebs solution was used inthe control experiments.

For the in vivo rat and mouse experiments, 5.5 ml of the 1 mg/ml stocksolution of CYT-1010 was prepared freshly every experimental day with20% HPCD dissolved in sterile distilled water. This milky whitemicro-suspension was shaken and sonicated. The vehicle was 20% HPCDdissolved in sterile distilled water. Further dilutions of the compoundfor the smaller administered doses were made with this vehicle, the 500μg/ml and 100 μg/ml concentrations looked opalescent and the lowerconcentrations were clear. In rats 0.1 ml/100 g, in mice 0.1 ml/10 gvolumes were administered i.v. 10 min before the induction of theinflammation.

Induction of Sensory Neuropeptide Release from Isolated Rat Trachea byElectrical Field Stimulation (EFS)

Experimental Model

Rats were exsanguinated in deep anaesthesia (sodium thiobarbital, 50mg/kg i.p.), then the whole trachea was removed and cleaned of fat andadhering connective tissues. Tracheae from two rats were placed into thesame 1.8 ml organ bath to obtain sufficient amount of released peptideand perfused (1 ml/min) with pH-(7.2) controlled oxygenized Krebssolution for 60 minutes (equilibration period) at 37° C. temperature.After discontinuation of the flow, the solution was changed three timesfor eight minutes to produce pre-stimulated, stimulated, post-stimulatedfractions.

Electrical field stimulation (40 V, 0.1 ms, 10 Hz for 120 s; 1200pulses) was performed to elicit neurotransmitter release at thebeginning of the second 8-minute period. Stimulation with 0.1 ms pulsewidth selectively activates very fast Na⁺ channels, which are onlypresent in the membrane of neural structures (Birmingham and Wilson,1963; Coburn and Tomita 1973; Szolcsányi and Barthó, 1982); therefore,it excites nerve endings without influencing other excitable cells inthe tracheae such as smooth muscle cells.

Protocol

Krebs solution was used in the pre-stimulated fraction for determiningthe basal CGRP outflow. During the stimulated and post-stimulatedfractions, the incubation medium contained the examined concentrationsof CYT-1010 (10, 100, 500, 1000 and 2000 nM) in separate experiments,and only one concentration was applied to the same tracheae to avoidneuropeptide depletion. In each group, 5 experiments were performed inparallel in 5 perfusion systems to provide n=5 data per group (10tracheae per group).

Animals

60 male Wistar rats (250-300 g) altogether were divided into 6experimental groups: Group 1: controls (n=10); Group 2: 10 nM CYT-1010(n=10); Group 3: 100 nM CYT-1010 (n=10); Group 4: 500 nM CYT-1010(n=10); Group 5: 1000 nM CYT-1010 (n=10); and Group 6: 2000 nM CYT-1010(n=10). The total study was performed on three experimental days, twogroups every occasion.

Investigational Technique: Measurement of CGRP and SP Concentrations byRadioimmunoassay

Calcitonin gene-related peptide (CGRP) and substance P (SP)concentrations were determined from 400-400 μl samples of organ fluid ofthe preparations by means of radioimmunoassay methods described inNemeth et al., 1996, 1998, 1999, 2006; Helyes et al., 1997, 2001, 2006;Borzsei et al., 2008). Each of these publications is incorporated hereinby reference in its entirety.

The released amount of the peptides CGRP and SP was calculated as fmolpeptide per mg wet tissue (trachea). The absolute release in response toEFS in each experiment was calculated by adding peptide release in thesecond and third 8-min fractions and then taking off the basal releasemeasured in the first (pre-stimulated) fraction. The detection limits ofthe RIA assays were 0.2 fmol/tube and 2 fmol/tube for CGRP and SP,respectively.

Induction of Acute Neurogenic Inflammation in the Mouse Ear by MustardOil

Experimental Model

Mice were anaesthesized with urethane (1.2 g/kg i.p.) to achieve along-lasting deep anaesthesia and minimize respiratory depression. Tenμl of 5% mustard oil dissolved in paraffin oil was smeared on both sidesof both ears. Mustard oil in this concentration selectively activatesTransient Receptor Potential A1 (TRPA1) on capsaicin-sensitivepeptidergic sensory nerves and induces the release of sensoryneuropeptides. The released CGRP and substance P (SP) in the innervatedarea evoke vasodilatation and plasma protein extravasation, collectivelycalled as acute neurogenic inflammation.

Protocol

CYT-1010 (1, 10, 100, 500 and 1000 μg/kg; 0.1 ml/10 g from the 0.1, 1,10, 50 and 100 μg/ml solutions) was administered i.v. 5 min before theinduction of the inflammation by mustard oil smearing. Mice in thecontrol group were treated with the same volume of the vehicle, 20%hydroxypropyl-β-cyclodextrin dissolved in sterile distilled water. Theexperimental protocol is also illustrated in FIG. 1.

Animals

There were 8 mice in each CYT-1010-treated experimental group, and 9 inthe vehicle-treated control group. 49 male CD1 mice (25-35 g) altogetherwere divided into 6 experimental groups: Group 1: vehicle-treatedcontrols (n=9); Group 2: 1 μg/kg CYT-1010 (n=8); Group 3: 10 μg/kgCYT-1010 (n=8); Group 4: 100 μg/kg CYT-1010 (n=8); Group 5: 500 μg/kgCYT-1010 (n=8); and Group 6: 1000 μg/kg CYT-1010 (n=8).

This study was undertaken in blocks with 12-13 mice per occasion. Thewhole set of data was obtained during 4 days. There were 2 or 3solvent-treated rats every day and the remaining 12 animals wererandomised to receive each treatment.

Investigational Technique: Measurement of Mouse Ear Swelling withMicrometry

The diameter of the ear was measured with an engineers' micrometerbefore the treatment and 4 times during the 3 h-examination period (20min, 1 h, 2 h and 3 h). Oedema was expressed in % compared to theinitial control values (Banvolgyi et al., 2004, 2005; Borzsei et al.,2008). Each of these publications is incorporated herein by reference inits entirety.

Induction of Acute Neurogenic Inflammation in the Paw Skin of the Rat byMustard Oil

Experimental Model

Both hindlegs of the rats were acutely denervated (the sciatic and thesaphenous nerves were cut 30 min before the induction of inflammation)under deep sodium thiopental (50 mg/kg i.p.) anaesthesia to avoidcentral reflexes. Acute neurogenic inflammation in the dorsal skin ofthe hindpaws was evoked by topical application of 1% mustard oil(allylisothiocianante) dissolved in paraffin oil. Mustard oil inconcentrations below 5% selectively stimulates TRPA1 ion channels oncapsaicin-sensitive peptidergic nerves and induces the release ofpro-inflammatory sensory neuropeptides such as CGRP and substance P,which cause vasodilation and plasma protein extravasation in theinnervated area.

Protocol

CYT-1010 (1, 10, 100 and 1000 μg/kg; 0.1 ml/100 g from the 1, 10, 100and 1000 μg/ml solutions) was administered i.v. 5 min before theinduction of the inflammation by mustard oil smearing. Rats in thecontrol group were treated with the same volume of the vehicle, 20%hydroxypropyl-β-cyclodextrin (dissolved in sterile distilled water). Theexperimental protocol is also illustrated in FIG. 2.

Animals

40 male Wistar rats (220-260 g) altogether were divided into 5experimental groups: Group 1: 8 vehicle-treated controls (n=8); Group 2:1 μg/kg CYT-1010 (n=8); Group 3: 10 μg/kg CYT-1010 (n=8); Group 4: 100μg/kg CYT-1010 (n=8); and Group 5: 1000 μg/kg CYT-1010 (n=8).

This study was undertaken in blocks with 10 rats per occasion. The wholeset of data was obtained during 4 days, and there were rats of eachgroup every day.

Investigational Technique: Measurement of Evans Blue Accumulation in thePaw Skin

Extravasation of plasma albumin was measured by the Evans blue leakagemethod. Evans blue (50 mg/kg) was injected i.v. and neurogenicinflammation was induced 10 min later. Rats were killed byexsanguination 20 min after mustard oil application. The skin of thehindpaws was removed and the extravasated dye was extracted withformamide for 72 h at room temperature for photometric determination at620 nm. The amount of the accumulated Evans blue, which quantitativelycorrelates with the intensity of plasma extravasation, was expressed asμg dye/g wet tissue (Szolcsányi and Barthó 1981; Helyes et al., 1997,2001, 2006). Each of these publications is incorporated herein byreference in its entirety.

Anesthesia

In the first series of the in vivo experiments of mice, ketamine (100mg/kg, i.p.) and xylazine (10 mg/kg, i.m.) were used for anesthesiaaccording to the conventional protocol. This time all the 10 mice diedpromptly, within 5 minutes after i.v. CYT-1010 injection, presumably dueto respiratory depression; The 2 vehicle-treated animals survived.Therefore, urethane (1.2 g/kg i.p.) anaesthesia was chosen in thismodel, and in the further studies all mice survived except one in the 1μg/kg dose group (this one has been replaced later to have the n=8 ineach group).

Statistical Analysis

Results are expressed as the mean±s.e.m. and analyzed for statisticalsignificance with one-way ANOVA followed by Dunnett post-hoc test whenthe data were compared to the control group. In the rat in vivo studies,multiple comparisons were also made with Bonferroni's modified t-test tocompare the effects of the different doses to each other. *p<0.05 wasconsidered to be significant. All individual data are tabulated inTables 3-5.

Following are examples that illustrate procedures for practicing theinvention. These examples should not be construed as limiting.

EXAMPLE 1 Effect of CYT-1010 on Electrical Field Stimulation-EvokedRelease of CGRP and SP from Isolated Rat Tracheae

This Example demonstrates that CTT-1010 inhibits release of theEFS-induced pro-inflammatory sensory neuropeptide CGRP and SP.

In the control experiments, the release of CGRP and SP increased from0.17±0.03 fmol/mg to 0.57±0.09 fmol/mg wet tissue and 1.54+0.085 fmol/mgto 2.38+0.15 fmol/mg wet tissue, respectively, in the second 8-minfraction, as a result of the electrical field stimulation. The peptiderelease values were still elevated in the third 8-fraction afterstimulation (CGRP: 0.33±0.04 fmol/mg; SP: 2.24+0.27 fmol/mg) (FIGS. 3,4). The absolute release in response to EFS in the second and thirdfractions after taking off the basal release was 0.56±0.09 fmol/mg forCGRP and 1.53+0.25 fmol/mg for SP.

Addition of CYT-1010 (10-2000 nM) to the second and third fractionssignificantly inhibited the stimulation-evoked CGRP and SP release, butconcentration-response relationship was only observed in the smallerconcentration range, between 10 and 500 nM for CGRP and 100-1000 nM forSP. In case of CGRP release, the maximal effect of 69.9% inhibition wasobtained with 500 nM CYT-1010, and the 1000 and 2000 nM concentrationsdid not increase further the inhibitory action (66.3% and 49.1%,respectively). However, the two highest concentrations abolished, andthe 100 and 500 nM significantly inhibited CGRP release in the third,post-stimulated fraction; while the 10 nM did not influence CGRP releasein the third, post-stimulated fraction. For SP outflow the maximalinhibition of 71.8% was observed in the presence of the 1000 nMconcentration, while 500 nM CYT-1010 evoked 64.3% inhibitory action.

When analyzing the experimental data as sigmoidal concentration-responsecurves via a non-linear least square curve fitting procedure, the EC₅₀calculated from the best fit values proved to be 40.6 nM for CGRP and34.0 nM for SP (FIGS. 5, 6).

TABLE 3 Release of CGRP into the incubation medium (fmol/mg wet tissue)in the pre-stimulated, stimulated and post-stimulated 8-min fractions %increase above Experiment 1 2 3 4 5 Mean S.E.M. baseline Control(vehicle) Before stimulation 0.123 0.191 0.105 0.250 0.201 0.174 0.0270.00 During stimulation 0.490 0.805 0.256 0.649 0.664 0.573 0.094 229.20After stimulation 0.386 0.349 0.200 0.297 0.436 0.334 0.040 91.66Absolute release 0.631 0.772 0.245 0.445 0.698 0.558 0.095 — CYT-1010 2microM Before stimulation 0.102 0.231 0.097 0.148 0.134 0.142 0024 0.00During stimulation 0.198 0.584 0.388 0.645 0.353 0.433 0.081 204.09After stimulation 0.200 0.161 0.133 0.018 0.167 0.136 0.031 −4.59Absolute release 0.192 0.284 0.326 0.366 0.253 0.284 0.030 — CYT-1010 1microM Before stimulation 0.262 0.135 0.108 0.108 0.102 0.143 0.030 0.00During stimulation 0.510 0.433 0.170 0.170 0.161 0.289 0.076 101.59After stimulation 0.386 0.162 0.130 0.130 0.121 0.186 0.051 29.82Absolute release 0.371 0.325 0.083 0.083 0.077 0.188 0.066 — CYT-1010500 nM Before stimulation 0.136 0.148 0.146 0.118 0.157 0.141 0.007 0.00During stimulation 0.162 0.301 0.304 0.192 0.241 0.240 0.028 70.52 Afterstimulation 0.137 0.206 0.267 0.224 0.213 0.210 0.021 48.89 Absoluterelease 0.028 0.212 0.279 0.181 0.140 0.168 0.042 — CYT-1010 100 nMBefore stimulation 0.243 0.129 0.128 0.138 0.183 0.164 0.022 0.00 Duringstimulation 0.586 0.281 0.526 0.390 0.554 0.468 0.057 184.81 Afterstimulation 0.284 0.135 0.267 0.231 0.226 0.229 0.026 39.26 Absoluterelease 0.384 0.159 0.538 0.344 0.415 0.368 0.061 — CYT-1010 10 nMBefore stimulation 0.220 0.250 0.146 0.256 0.226 0.219 0.020 0.00 Duringstimulation 0.600 0.420 0.299 0.308 0.630 0.451 0.070 105.75 Afterstimulation 0.602 0.730 0.193 0.204 0.528 0.451 0.108 105.75 Absoluterelease 0.762 0.650 0.201 0.001 0.706 0.464 0.153 —

TABLE 4 SP Release into the incubation medium (fmol/mg wet tissue) inthe pre-stimulated, stimulated and post-stimulated 8-min fractions %increase above Experiment 1 2 3 4 5 Mean S.E.M. baseline Control(vehicle) Before stimulation 1.332 1.567 1.473 1.839 1.506 1.541 0.0850.00 During stimulation 1.990 2.386 2.219 2.903 2.398 2.379 0.150 54.35After stimulation 1.921 2.907 1.814 2.883 1.654 2.236 0.272 45.05Absolute release 1.267 2.159 1.087 2.108 1.040 1.532 0.249 — CYT-1010 2microM Before stimulation 2.406 1.582 2.020 1.132 1.867 1.801 0.214 0.00During stimulation 3.130 2.398 2.334 2.078 4.132 2.814 0.373 56.23 Afterstimulation 2.348 1.531 2.246 1.543 1.944 1.922 0.171 6.72 Absoluterelease 0.666 0.765 0.540 1.357 2.342 1.134 0.333 — CYT-1010 1 microMBefore stimulation 1.398 1.806 1.860 1.412 0.805 1.456 0.189 0.00 Duringstimulation 1.558 1.948 1.992 1.422 1.331 1.650 0.136 13.32 Afterstimulation 1.663 1.966 1.892 1.551 1.397 1.694 0.106 16.32 Absoluterelease 0.425 0.302 0.164 0.149 1.118 0.432 0.179 —

EXAMPLE 2 Effect of CYT-1010 on Acute Neurogenic Oedema of the Mouse Ear

This Example demonstrates that CYT-1010 significantly inhibits mustardoil-induced acute neurogenic oedema.

In the control, vehicle-treated group ear thickness increased from312.2±4.8 μm to 348.3±7.7 μm 20 min after topical application of 5%mustard oil (11.65±1.98% swelling). This increased further to 37.83±0.83mm by 1 h and remained relatively unchanged until 3 hours.

At 1 hour, the 1, 10 and 100 μg/kg doses exerted a significantanti-edema action, although 20 minutes post induction, none of theexamined CYT-1010 doses, which were administered i.v. 5 min before theinduction of the inflammation, significantly inhibited mustardoil-induced ear swelling. Furthermore, the inhibitory action of 10 and100 μg/kg CYT-1010 proved to be statistically significant at both latertime points as well. The 500 and 1000 μg/kg dose did not influence earswelling. Dose-response correlation was not observed in this model (FIG.7). The AUC value calculated on the basis of the time course ofpercentage ear swelling was 55.29±4.91 units in the control,vehicle-treated group. In comparison to the control, the correspondingdata in mice treated with CYT-1010 (1, 10, 100, 500 and 1000 μg/kg) were28.99±4.60 (P<0.05), 28.15±3.42 (P<0.01), 26.55±5.51 (P<0.01),52.24±9.70 (NS) and 37.25±5.72 (NS), respectively.

EXAMPLE 3 Effect of CYT-1010 on Mustard Oil-Induced Plasma ProteinExtravasation in the Rat Skin

This Example demonstrates that CYT-1010 significantly inhibits mustardoil-induced plasma protein extravasation.

Specifically, all of the examined CYT-1010 doses (1, 10, 100 and 1000μg/kg, i.v.) exerted significant inhibition on 1% mustard oil-inducedneurogenic plasma protein extravasation in the dorsal skin of the rathindpaw detected by the Evans blue leakage technique. However, nodose-response correlation was observed in this model. The smallestapplied dose had the greatest, 52.83% inhibitory action, while the threehigher doses similarly diminished plasma protein extravasation by27.89%, 36.76% and 29.90%, respectively. The inhibitory action of 1μg/kg CYT-1010 was significantly greater than those of the 10 and 100μg/kg doses (FIG. 6).

TABLE 5 Evans blue leakage in the rat hindpaw skin (μg/g wet skin) inresponse to topical administration of 1% mustard oil Control CYT-1010i.v. (vehicle) 1 μg/kg 10 μg/kg 100 μg/kg 1000 μg/kg 1 left 129.10 48.5291.18 52.19 76.56 1 right 89.03 44.06 61.48 49.21 54.68 2 left 99.6333.73 77.24 80.28 63.41 2 right 92.07 63.04 87.67 71.20 55.85 3 left86.96 42.27 79.89 54.84 66.07 3 right 74.19 3.46 77.63 83.92 85.40 4left 80.28 41.04 87.14 49.09 79.34 4 right 102.49 31.69 108.49 54.86154.03 5 left 103.06 46.87 73.59 59.74 74.05 5 right 129.17 49.44 65.6988.54 88.00 6 left 97.34 47.62 78.05 56.89 62.92 6 right 120.26 77.5194.66 75.00 55.80 7 left 109.70 53.72 36.25 56.81 67.62 7 right 118.1148.91 51.07 63.41 57.47 8 left 85.49 59.80 41.10 68.93 37.45 8 right87.80 65.26 46.38 49.74 46.10 Mean 100.29 47.31 72.34 63.42 70.30 SEM4.24 4.14 5.13 3.25 6.55

As demonstrated in Examples 1-3, electrical field stimulation (EFS)evoked more than 3-fold elevation of CGRP release compared to its basaloutflow. The total absolute peptide release was decreased by 70% and 66%in response to adding CYT-1010 into the organ bath during and afterstimulation in 500 nM and 1 μM concentrations, respectively. Also,electrically-induced SP release was 54% in the stimulated and 45% in thepost-stimulated fractions, respectively. The total release wassignificantly diminished by 64% and 72% in the presence of 500 nM and 1μM CYT-1010. When analyzing the data as sigmoidal concentration-responsecurves via a non-linear least square curve fitting procedure, the EC₅₀calculated from the best fit values proved to be 40.6 nM for CGRP and34.0 nM for SP. A concentration-response relationship was observed inthe concentration range of 10-500 nM for CGRP and 100-1000 nM for SP.

Mustard oil-induced acute neurogenic ear swelling of the mouse wassignificantly reduced by 1 μg/kg, 10 μg/kg and 100 μg/kg i.v. doses ofCYT-1010. A similar significant anti-inflammatory effect was found onmustard oil-evoked plasma protein extravasation in the rat skin by i.v.pretreatment with 1-1000 μg/kg CYT-1010; the lowest dose exerted thegreatest effect, almost 60% inhibitory action.

Based on these results it can be concluded that CYT-1010 effectivelyattenuates neurogenic inflammatory responses in which the inhibition ofsensory neuropeptide release is likely to be involved. Since this typeof inflammation is not affected by the presently available non-steroidalanti-inflammatory/analgesic drugs and high doses of corticosteroidsinduce only a moderate inhibition, this stable and highly potent EM-1analog provides a novel treatment of inflammatory conditions where painand nociceptor activation play a pivotal role.

EXAMPLE 4 Effects of Filtered and Non-Filtered CYT-1010 on MustardOil-Induced Acute Neurogenic Ear Swelling in Mice

This Example demonstrates that filtration of CYT-1010 moderatelydecreases the inhibitory action of CYT-1010. This inhibitory effect wasmarkedly less in comparison with filtered vehicle controls.

Materials and Methods

CYT-1010 (synthetic cyclized endomorphin-1 analog; FP014; Lot #:080811-R2; Bottle H22; MW:684), was purchased from AmbioPharm, Inc.(North Augusta, S.C. 29812). Additional materials include:2-hydroxypropyl-βcyclodextrin (HPCD; Batch No. L-36/07), purchased fromCyclolab Ltd., Budapest, Hungary; urethane, purchased from Spektrum 3D,Debrecen, Hungary; allylisothiocyanate (mustard oil), paraffin oil,purchased from Szkarabeusz Ltd., Pécs, Hungary, and sterile syringefilters (PVDF; 0.2 μm; TR-200507; Lot. 134831), purchased from OlimPeak,Teknokroma, Barcelona, Spain.

Preparation of Solutions and Suspensions

Stock solution of the 100 μg/ml concentration of CYT-1010 (5.5 ml) wasprepared fresh every experimental day with 20%hydroxypropyl-βcyclodextrin (HPCD) dissolved in sterile distilled water,shaken properly and sonicated. The vehicle was 20% HPCD dissolved insterile distilled water. Further dilutions of the compound for thesmaller administered doses were made with this vehicle. The appliedconcentrations were filtered through a 0.2 μm sterile teflon filter.This 100 μg/ml solution was opalescent directly after preparation, butit cleared up 1 hour later at C. The lower concentrations looked clearimmediately. Non-filtered solutions were administered in the sameconcentrations in the parallel groups for comparison. Three doses ofboth the filtered and non-filtered CYT-1010 solutions (1, 10 and 100μg/kg; 0.1 ml/10 g from the 0.1, 1 and 10 μg/ml solutions) wereadministered i.v. 5 min before the induction of the inflammation.Animals of the control group were treated with the same volume of theHPCD after or without the same filtration procedure.

Mustard Oil-induced Acute Neurogenic Inflammation in the Mouse Ear

Experimental Model

Mice were anaesthesized with urethane (1.2 g/kg i.p.) to achieve along-lasting deep anaesthesia and minimize respiratory depression. Tenμl of 5% mustard oil dissolved in paraffin oil was smeared on both sidesof both ears. Mustard oil in this concentration selectively activatesTransient Receptor Potential A1 (TRPA1) on capsaicin-sensitivepeptidergic sensory nerves and induces the release of sensoryneuropeptides (Banvolgyi et al., 2004). The released calcitoningene-related peptide (CGRP) and tachykinins such as substance P (SP) inthe innervated area evoke vasodilatation and plasma proteinextravasation collectively called acute neurogenic inflammation (Helyeset al., 1997, 2001, 2006; Borzsei et a., 2008).

Protocol

CYT-1010 (1, 10 and 100 μg/kg; 0.1 ml/10 g from the 0.1, 1, 10 μg/mlsolutions) was administered i.v. 5 min before the induction of theinflammation by mustard oil smearing. Mice in the control group weretreated with the same volume of the vehicle, 20% HPCD dissolved insterile distilled water

There were 8 mice in each experimental group, and 9 in the 1 μg/kgfiltered CYT-1010 dose group CYT-1010-treated vehicle-treated controlgroup.

Animals

65 male CD1 mice (25-35 g) were divided into 8 experimental groups:Group 1: non-filtered vehicle-treated controls (n=8); Group 2:non-filtered 1 μg/kg CYT-1010 (n=8); Group 3: non-filtered 10 μg/kgCYT-1010 (n=8); Group 4: non-filtered 100 μg/kg CYT-1010 (n=8); Group 5:filtered vehicle-treated controls (n=8); Group 6: filtered 1 μg/kgCYT-1010 (n=9); Group 7: filtered 10 μg/kg CYT-1010 (n=8); and Group 8:filtered 100 μg/kg CYT-1010 (n=8).

This study was undertaken in blocks with 16-17 mice per occasion. Thewhole set of data was obtained during 4 days (12, 13, 16, 22 Apr. 2010).There were 2 or 3 solvent-treated rats every day and the remaining 12animals were randomised to receive each treatment.

Investigational Technique Measurement of Mouse Ear Swelling withMicrometry

The diameter of the ear was measured with an engineers' micrometerbefore the treatment and 4 times during the 3 h-examination period (20min, 1 h, 2 h and 3 h). Edema was expressed in % compared to the initialcontrol values (Banvolgyi et al., 2004; Borzsei et al., 2008). Thecompound was administered i.v. 5 min before mustard oil smearing in 3doses (1, 10, 100 μg/kg) in separate groups after sterile filtration orwithout filtration. Animals of the control group were treated with thesame volume of the vehicle

Statistical Analysis

Results are expressed as the mean±s.e.m. Statistical significance wasperformed with one-way ANOVA followed by Dunnett's post-hoc test(modified t-test) when the data of the CYT-1010-treated groups werecompared to the respective vehicle-treated controls at certain timepoints. The areas under the time-dependent percentage ear swellingcurves (AUC) were also calculated and analyzed with the same statisticsto compare the whole edema responses of the CYT-1010-treated mice duringthe 3 h-experimental period to the respective control group. *p<0.05 wasconsidered to be significant. Microsoft Excel was used for storing theindividual data and preparing the worksheets, GrapPadPrism5 forstatistical analysis and Origin 7.0 for making the graphs.

Ethics

All experimental procedures were carried out according to the1998/XXVIII Act of the Hungarian Parliament on Animal Protection andConsideration Decree of Scientific Procedures of Animal Experiments(243/1988) and complied with the recommendations of the InternationalAssociation for the Study of Pain and the Helsinki Declaration. Thestudies were approved by the Ethics Committee on Animal Research of PécsUniversity according to the Ethical Codex of Animal Experiments andlicensee was given (licensee No.: BA 02/2000-11-2006).

Effect of Non-Filtered CYT-1010 on Acute Neurogenic Edema of the MouseEar

In the control, non-filtered vehicle-treated group ear thicknessincreased from 312.50±4.70 μm to 361.25±6.05 μm 20 min after topicalapplication of 5% mustard oil (15.94±2.51% swelling). This remainedrelatively stable during 2 hours, then started to minimally decrease.The 10 μg/kg and 100 μg/kg CYT-1010 i.v. doses without filtrationadministered 5 min before the induction of the inflammationsignificantly inhibited mustard oil-induced ear swelling 20 min later,and these inhibitory actions were observed until the 2 hour time point.Although the edema-decreasing action of 10 μg/kg CYT-1010 was notstatistically significant at 1 h with the Dunnett's post test we usedfor data analysis, the inhibitory tendency was clearly seen. The 1 μg/kgdose did not significantly diminish ear swelling, although a minimalreduction was observed in the first 2 hours. Dose-response correlationwas not found in this model (FIG. 9). The AUC value calculated on thebasis of the time course of percentage ear swelling was 44.02±6.97 unitsin the control, HPCD vehicle-treated group. In comparison to thecontrol, the corresponding data in mice treated with CYT-1010 (1, 10 and100 μg/kg) were 40.33±5.32 (NS), 24.21±2.75 (P<0.05), 21.71±1.72(P<0.01), respectively.

Effect of Filtered CYT-1010 on Acute Neurogenic Edema of the Mouse Ear

In the control, filtered HPCD vehicle-treated group ear thicknessincreased from 294.17±4.17 μm to 339.17±7.63 μm 20 min after topicalapplication of 5% mustard oil (17.85±2.01% swelling). This remainedalmost unchanged throughout the 3 hour-examination period. The 10 μg/kgand 100 μg/kg doses of filtered CYT-1010 solutions administered i.v. 5min before the induction of the inflammation inhibited significantlymustard oil-induced ear swelling 20 min later, but statisticallysignificant anti-edema effect compared to the vehicle-treated controlgroup was only observed for the 10 μg/kg dose at the 3 h time point.However, the inhibitory actions of the filtered solution compared to therespective concentrations of the non-filtered compounds was notsignificantly different. The filtered 1 μg/kg dose did not influence earswelling. Similarly to the actions of the non-filtered compound,dose-response correlation was not observed in this experimental serieseither (FIG. 10). The AUC value calculated on the basis of the timecourse of percentage ear swelling was 50.04±6.96 units in the control,filtered HPCD vehicle-treated group. In comparison to the control, thecorresponding data in mice treated with the filtered CYT-1010 (1, 10 and100 μg/kg) were 51.47±6.93 (NS), 33.68±3.79 (NS), and 33.97±5.62 (NS),respectively.

In this Example 4, mustard oil-induced acute neurogenic ear swelling ofthe mouse was significantly reduced by 10 μg/kg and 100 μg/kg doses ofCYT-1010 injected i.v. without filtration. The extent of the inhibitionwas about 50% in both cases, there was no dose-response correlation. Aminimal inhibitory tendency was also observed in the 1 μg/kg dose in thefirst 2 hours. The HPCD vehicle after filtrating through a 0.2 μmsterile filter did not show any difference compared to the non-filteredcontrols. Administration of the two higher CYT-1010 doses afterfiltration exerted moderate inhibition. Compared to the respectivevehicle-treated controls statistically significant differences were onlyobserved at the 20 min time point and at 3 h in case of the 10 μg/kgdose. Analysing the areas under the curves did not show any significantdifferences between any group, although a clear inhibitory tendency wasobserved for both the 10 and 100 CYT-1010 treatment. The filtered 1μg/kg dose did not have any effect.

Based on these results it can be concluded that filtration moderatelydecreases the inhibitory action of CYT-1010 in this model. Although thiseffect was not significantly smaller than that of the respectivenon-filtered formulations, it was markedly less in comparison with thefiltered vehicle controls.

All patents, patent applications, provisional applications, andpublications referred to or cited herein are incorporated by referencein their entirety, including all figures and tables, to the extent theyare not inconsistent with the explicit teachings of this specification.

The terms “a” and “an” and “the” and similar referents as used in thecontext of describing the invention are to be construed to cover boththe singular and the plural, unless otherwise indicated herein orclearly contradicted by context.

Recitation of ranges of values herein are merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range, unless otherwise indicated herein, and eachseparate value is incorporated into the specification as if it wereindividually recited herein. Unless otherwise stated, all exact valuesprovided herein are representative of corresponding approximate values(e.g., all exact exemplary values provided with respect to a particularfactor or measurement can be considered to also provide a correspondingapproximate measurement, modified by “about,” where appropriate).

The use of any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise indicated. No language in the specification should beconstrued as indicating any element is essential to the practice of theinvention unless as much is explicitly stated.

The description herein of any aspect or embodiment of the inventionusing terms such as “comprising”, “having”, “including” or “containing”with reference to an element or elements is intended to provide supportfor a similar aspect or embodiment of the invention that “consists of”,“consists essentially of”, or “substantially comprises” that particularelement or elements, unless otherwise stated or clearly contradicted bycontext (e.g., a composition described herein as comprising a particularelement should be understood as also describing a composition consistingof that element, unless otherwise stated or clearly contradicted bycontext).

It should be understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to be included within the spirit and purview of thisapplication.

We claim:
 1. A method for treating a human subject to reduce neurogenicinflammation, wherein said method comprises administering,intravenously, to a human subject in need of treatment of neurogenicinflammation, an effective amount of the acetate salt of SEQ ID NO:13.2. The method, according to claim 1, further comprising administering ananti-inflammatory agent selected from the group consisting of asteroidal compound, hydrocortisone, a non-steroidal anti-inflammatorycompound, acetylsalicylic acid (aspirin), ibuprofen, acetaminophen, andindomethacin, wherein the anti-inflammatory agent is administeredbefore, during, or after the administration of said peptide or saltthereof.